Stabilizing and Accelerating Secondary Flow in Ultralong Spiral Channel for High-Throughput Cell Manipulation.

Efficient cell manipulation is essential for numerous applications in bioanalysis and medical diagnosis. However, the lack of stability and strength in the secondary flow, coupled with the narrow range of practical throughput, severely restricts the diverse applications. Herein, we present an innovative inertial microfluidic device that employs a spiral channel for high-throughput cell manipulation. Our investigation demonstrates that the regulation of Dean-like secondary flow in the microchannel can be achieved through geometric confinement. Introducing ordered microstructures into the ultralong spiral channel (>90 cm) stabilizes and accelerates the secondary flow among different loops. Consequently, effective manipulation of blood cells within a wide cell throughput range (1.73 × 108 to 1.16 × 109 cells/min) and cancer cells across a broad throughput range (0.5 × 106 to 5 × 107 cells/min) can be achieved. In comparison to previously reported technologies, our engineering approach of stabilizing and accelerating secondary flow offers specific performance for cell manipulation under a wide range of high-throughput manner. This engineered spiral channel would be promising in biomedical analysis, especially when cells need to be focused efficiently on large-volume liquid samples.

Fascin-1 Promotes Cell Metastasis through Epithelial-Mesenchymal Transition in Canine Mammary Tumor Cell Lines.

Canine mammary tumors (CMTs) are the most common type of tumor in female dogs. In this study, we obtained a metastatic key protein, Fascin-1, by comparing the proteomics data of in situ tumor and metastatic cell lines from the same individual. However, the role of Fascin-1 in the CMT cell line is still unclear. Firstly, proteomics was used to analyze the differential expression of Fascin-1 between the CMT cell lines CHMm and CHMp. Then, the overexpression (CHMm-OE and CHMp-OE) and knockdown (CHMm-KD and CHMp-KD) cell lines were established by lentivirus transduction. Finally, the differentially expressed proteins (DEPs) in CHMm and CHMm-OE cells were identified through proteomics. The results showed that the CHMm cells isolated from CMT abdominal metastases exhibited minimal expression of Fascin-1. The migration, adhesion, and invasion ability of CHMm-OE and CHMp-OE cells increased, while the migration, adhesion, and invasion ability of CHMm-KD and CHMp-KD cells decreased. The overexpression of Fascin-1 can upregulate the Tetraspanin 4 (TSPAN4) protein in CHMm cells and increase the number of migrations. In conclusion, re-expressed Fascin-1 could promote cell EMT and increase lamellipodia formation, resulting in the enhancement of CHMm cell migration, adhesion, and invasion in vitro. This may be beneficial to improve female dogs' prognosis of CMT.

Dietary supplementation of osthole and icariin improves the production performance of laying hens by promoting follicular development.

Osthole (Ost) and icariin (Ica) are extracted from traditional Chinese medicine Cnidium monnieri and Epimedii Folium, respectively, and both exhibit estrogen-like biological activity. This study aimed to determine the efficacy and safety of combining Ost with Ica on the production performance of laying hens and to explore their possible mechanisms. The production performance, egg quality, residues of Ost and Ica in eggs, serum reproductive hormone levels, expression of ovarian reproductive hormone receptor, proliferation of granulosa cells in small yellow follicles (SYF), and progesterone secretion in large yellow follicles (LYF) related genes and proteins expression were detected. The results showed that adding 2 mg/kg Ost + 2 mg/kg Ica to the feed increased the laying rate, average egg weight, Haugh unit, and protein height of laying hens. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and progesterone (P4) levels increased, and the expression of ovarian estrogen receptor (ER), follicle-stimulating hormone receptor (FSHR), and progesterone receptor (PGR) mRNA was up-regulated. Additionally, the mRNA and protein levels of steroidogenesis acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) increased in LYF. Furthermore, mRNA and protein levels of proliferating cell nuclear antigen (PCNA), cyclin E1, and cyclin A2 were up-regulated in SYF. The residues of Ost and Ica in egg samples were not detected by high-performance liquid chromatography (HPLC). In conclusion, dietary supplementation of Ost and Ica increased granulosa cells proliferation in SYF and increased P4 secretion in granulosa cells of LYF, ultimately improving the production performance of laying hens.

Baicalin ameliorates high fat diet-induced nonalcoholic fatty liver disease in mice via adenosine monophosphate-activated protein kinase-mediated regulation of SREBP1/Nrf2/NF-κB signaling pathways.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease around the world, imposing severe threats on human health. Unfortunately, no clinically approved drugs are available for use as yet. Baicalin (BA) is reported to have hepatoprotective effects, and it is not clear whether BA can treat NAFLD and how. Here, a high-fat diet (HFD)-induced NAFLD mouse model was established to explore the protective roles and mechanisms of BA against HFD-induced NAFLD. Physiochemical results showed that BA exhibited significantly protective effects against HFD-induced NAFLD in mice. Liver transcriptomic analysis revealed that BA attenuated HFD-induced NAFLD via activating AMPK pathway, which was confirmed by the AMPK inhibitor Compound C. Additionally, the expression changes of AMPK downstream genes demonstrated that BA exerted ameliorative effects against NAFLD through AMPK-mediated inhibition of SREBP1 and NF-κB pathways, and activation of Nrf2 pathway. Taken together, our study reveals the protective roles of BA against HFD-caused NAFLD through AMPK-mediated modulation of SREBP1/Nrf2/NF-κB pathways, suggesting that BA has potential drug development implications. Most importantly, our study creates a paradigm through the combination of molecular biology and bioinformatics for further studies of action mechanisms of biomolecules combating diseases.

Matrine Targets BTF3 to Inhibit the Growth of Canine Mammary Tumor Cells.

The canine mammary tumor model is more suitable for studying human breast cancer, and the safety concentrations of matrine and the biotin-labeled matrine probe were determined in canine primary mammary epithelial cells, and then selected canine mammary tumor cell lines CHMm and CHMp were incubated with matrine, and cell viability was detected by CCK-8. The biotin-labeled matrine probe was used to pull-down the targets of matrine in canine mammary tumor cells, and the targets were screened in combination with activity-based protein profiling (ABPP) and Genecards database, and verified by qPCR and western blot. The results showed that the maximum non-cytotoxic concentrations of matrine and biotin-labeled matrine probe in canine primary mammary epithelial cells were 250 µg/mL and 500 µg/mL, respectively. Matrine and biotin-labeled matrine probe had a proliferation inhibitory effect time-dependently on CHMm and CHMp cells within a safe concentration range, and induced autophagy in cells. Then BTF3 targets were obtained by applying ABPP and Genecards screening. Cellular thermal shift assay (CETSA) findings indicated that matrine could increase the heat stability of BTF3 protein. Pull-down employing biotin-labeled matrine probe with CHMm and CHMp cell lysates revealed that BTF3 protein was detected in the biotin-labeled matrine probe group and that BTF3 protein was significantly decreased by the addition of matrine. The qPCR and western blot findings of CHMm and CHMp cells treated with matrine revealed that matrine decreased the expression of the BTF3 gene and protein with the extension of the action time, and the impact was more substantial at the protein level, respectively.

A novel strategy for optimal component formula of anti-PRRSV from natural compounds using tandem mass tag labeled proteomic analyses.

BACKGROUND: Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most important porcine viral diseases which have been threatening the pig industry in China. At present, most commercial vaccines fail to provide complete protection because of highly genetic diversity of PRRSV strains. This study aimed to optimize a component formula from traditional Chinese medicine(TCM)compounds with defined chemical characteristics and clear mechanism of action against PRRSV. METHODS: A total of 13 natural compounds were screened for the anti-PRRSV activity using porcine alveolar macrophages (PAMs). Three compounds with strong anti-PRRSV activity were selected to identify their potential protein targets by proteomic analysis. The optimal compound formula was determined by orthogonal design based on the results of proteomics. MTT assay was used to determine the maximum non-cytotoxic concentration (MNTC) of each compound using PAMs. QPCR and western blot were used to investigate the PRRSV N gene and protein expression, respectively. The Tandem Mass Tag (TMT) technique of relative quantitative proteomics was used to detect the differential protein expression of PAMs treated with PRRSV, matrine (MT), glycyrrhizic acid (GA) and tea saponin (TS), respectively. The three concentrations of these compounds with anti-PRRSV activity were used for orthogonal design. Four formulas with high safety were screened by MTT assay and their anti-PRRSV effects were evaluated. RESULTS: MT, GA and TS inhibited PRRSV replication in a dose-dependent manner. CCL8, IFIT3, IFIH1 and ISG15 were the top four proteins in expression level change in cells treated with MT, GA or TS. The relative expression of IFIT3, IFIH1, ISG15 and IFN-ß mRNAs were consistent with the results of proteomics. The component formula (0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 µg/mL TS) showed synergistic anti-PRRSV effect. CONCLUSIONS: The component formula possessed anti-PRRSV activity in vitro, in which the optimal dosage on PAMs was 0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 µg/mL TS. Compatibility of the formula was superposition of the same target with GA and TS, while different targets of MT. IFN-ß may be one of the targets of the component formula possessed anti-PRRSV activity.

Damage to intestinal barrier integrity in piglets caused by porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome (PRRS) induces respiratory disease and reproductive failure accompanied by gastroenteritis-like symptoms. The mechanism of intestinal barrier injury caused by PRRSV infection in piglets has yet to be investigated. An in vivo PRRSV-induced model was established in 30-day-old piglets by the intramuscular injection of 2 mL of 104 TCID50/mL PRRSV for 15 days. Observations of PRRSV replication and histology were conducted in the lungs and intestine, and goblet cell counts, relative MUC2 mRNA expression, and tight junction protein, proinflammatory cytokine, TLR4, MyD88, IκB and p-IκB expression were measured. PRRSV replicated in the lungs and small intestine, as demonstrated by absolute RT-qPCR quantification, and the PRRSV N protein was detected in the lung interstitium and jejunal mucosa. PRRSV infection induced both lung and gut injury, markedly decreased villus height and the villus to crypt ratio in the small intestine, and obviously increased the number of goblet cells and the relative expression of MUC2 mRNA in the jejunum. PRRSV infection aggravated the morphological depletion of tight junction proteins and increased IL-1ß, IL-6, IL-8 and TNF-α expression by activating the NF-κB signalling pathway in the jejunum. PRRSV infection impaired intestinal integrity by damaging physical and immune barriers in the intestine by inducing inflammation, which may be related to the regulation of the gut-lung axis. This study also provides a new hypothesis regarding the pathogenesis of PRRSV-induced diarrhoea.

The combined usage of Matrine and Osthole inhibited endoplasmic reticulum apoptosis induced by PCV2.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an important and common DNA virus that infect pig and can cause immunosuppression and induce apoptosis in the infected cells. To escape the host immune system, PCV2 constantly builds up complex mechanisms or mutates genes, and that is why it is difficult to eradicate complex PCV2 infection by relying on vaccines and single compound. At present, there is few literature reports on the effective prevention and treatment of PCV2 infection by a combination of two or more compounds. Previously, we have demonstrated the anti-PCV2 effect of Matrine in vitro, but its mechanism has not been further evaluated. Literatures have proven that Osthole has a variety of pharmacological activities, and we tested the ability of Osthole to inhibit PCV2 replication in cell culture. Therefore, this study explored the synergistic antiviral effect of Matrine combined with Osthole and their synergistic anti-apoptotic mechanism. RESULTS: Osthole alone had an anti-PCV2 effect, and then its synergistic anti-PCV2 effect of Osthole and Matrine was better than that of Matrine or Osthole alone as demonstrated by qRT-PCR, IFA and Western blotting results. The anti-apoptotic mechanism of these two compounds by inducing the PERK pathway by PCV2 was elucidated through Annexin V-FITC/PI, JC-1 and Western blotting. Matrine and Osthole combination could inhibit the expression of Cap in Cap-transfected PK-15 cells, thus inhibiting Cap-induced PERK apoptosis. Ribavirin was used as a positive control. CONCLUSIONS: The combination of Osthole and Matrine had the synergistic effect of anti-PCV2 infection by directly inhibiting the expression of PCV2 Cap protein. The combination of these two compounds also inhibited PERK apoptosis induced by PCV2 Cap protein, possibly by regulating the level of GRP78. The results formed a base for further studies on the mechanism of anti-PCV2 in vivo using Matrine and Osthole combination and developing new anti-PCV2 compounds with Cap and GRP78 as therapeutic targets.

Recombinant porcine NK-lysin inhibits the invasion of hepatocellular carcinoma cells in vitro.

The therapeutics having ability to target cancer cells specifically and exhibit nominal cytopathic effect on normal healthy cells are highly significant for cancer therapeutic applications. Recombinant porcine natural killer lysin (rpNK-lysin) has proven cationic anti-bacterial and anti-tumor peptide. Herein, we report its anti-invasion and anti-metastasis effects on hepatocellular carcinoma (HCC) cells in vitro. We first investigate the maximum non-toxic concentration (MNTC) of rpNK-lysin for the normal hepato cells (L-02). Using MNTC rpNK-lysin, we explore anti-proliferative, anti-adhesive, anti-invasive and anti-metastatic effect of rpNK-lysin on three different HCC cells lines (SMMC-7721, 97-H and HepG2) through MTT, wound-healing, adhesion and invasion assay along with mRNA and protein expression. The results reveal that rpNK-lysin has potential to specifically inhibit HCC cells growth in a dose and time-dependent manner with a little cytopathic effect on the L-02 cells, effectively reduce migration, adhesion and invasion ability of HCC cells. rpNK-lysin significantly reduce Fascin1 expression, which subsequently decrease ß-catenin expression and metaloproteinases (MMP-2 and MMP9). This study suggest that MNTC rpNK-lysin has an anti-invasion and anti-metastasis effect on HCC cells in vitro through inhibition of Fascin 1 expression which regulates Wnt/ß-catenin signaling pathway by inducing ß-catenin degradation and subsequently results in suppression of MMP-2 and MMP9 expression.

Matrine inhibits IL-1ß secretion in primary porcine alveolar macrophages through the MyD88/NF-κB pathway and NLRP3 inflammasome.

Our previous studies demonstrated that matrine directly acts on the replication process of porcine reproductive and respiratory syndrome virus (PRRSV). Matrine inhibits viral replication and is also associated with the NF-κB signalling pathway. These results suggest that matrine has antiviral and anti-inflammatory effects. However, the specific anti-inflammatory mechanism of matrine is still unclear. In this study, we investigated the anti-IL-1ß mechanism of matrine, as IL-1ß is a major inflammatory cytokine, in porcine alveolar macrophages (PAMs) stimulated with 4 µg PRRSV 5'-untranslated region (UTR) RNA and 1 µg/mL LPS. After 5'UTR RNA and LPS co-stimulation of PAMs for 12 h, the expression of IL-1ß, IL-6, IL-8 and TNF-α was significantly increased. The results also showed that co-stimulation induced the expression of MyD88, and activated the NF-κB signalling pathway and NLRP3 inflammasome. Furthermore, matrine treatment downregulated MyD88, NLRP3 and caspase-1 expression, inhibited ASC speck formation, suppressed IκBα phosphorylation, and interfered with the translocation of NF-κB from the cytoplasm to the nucleus. These results suggest that matrine plays an important role in PAMs co-stimulated with PRRSV 5'UTR RNA and LPS via its effect on NF-κB and the NLRP3 inflammasome. These findings lay the foundation for the exploration of the clinical application of matrine in PRRSV disease.

Anti-Invasion and Antimetastatic Effects of Porcine Recombinant NK-lysin on SMMC-7721 Human Hepatocellular Carcinoma Cells.

The high invasion and metastasizing abilities of hepatocellular carcinoma (HCC) are the primary reasons for the high mortality rate of patients. Therefore, identification of agents to inhibit invasion and metastasis is very important for treatment of HCC. We analyzed the anti-invasion and antimetastatic effects of porcine recombinant NK-lysin, which was designed and expressed in vitro by our research group, on SMMC-7721 hepatocellular carcinoma cells via wound-healing assays, adhesion assays, invasion assays, real-time polymerase chain reaction (PCR), and Western blot analysis. MTT assay results indicated that NK-lysin inhibited the growth of SMMC-7721 cells in a dose- and time-dependent manner. NK-lysin reduced the ability of cell migration, adhesion, and invasion. Based on gene and protein expression analysis, NK-lysin decreased ß-catenin and MMP-2 expression. These results suggested that NK-lysin has anti-invasion and antimetastatic effects on hepatocellular carcinoma cells in vitro by reducing the level of the ß-catenin and MMP-2.

Suppression of the Wnt signaling pathway may contribute to the inhibition of proliferation of human hepatocellular carcinoma SMMC-7721 cells by esculetin.

The aims of the present study were to investigate the effects of esculetin on the proliferation of human hepatocellular carcinoma SMMC-7721 cells and to determine the underlying mechanism behind this activity. An MTT assay was used to assess cell proliferation, reverse transcription-quantitative polymerase chain reaction was used to determine the relative mRNA expression levels of ß-catenin, c-Myc and cyclin D1, and western blot analysis was utilized to determine the levels of the associated proteins. Compared with the dimethyl sulfoxide control, esculetin reduced the cell viability of SMMC-7721 and HL-7702 cells in a dose- and time-dependent manner. Treatment of SMMC-7721 cells with esculetin resulted in downregulation of the mRNA and protein levels of ß-catenin, c-Myc and cyclin D1. Esculetin increased the phosphorylation of ß-catenin at Ser33/Ser37/Thr41 and inhibited the proliferation of human hepatoma SMMC-7721 cells by suppressing the Wnt signaling pathway. The results of the present study suggest that esculetin inhibited the Wnt/ß-catenin signaling pathway in SMMC-7721 cells and may have potential as an effective anti-cancer drug, acting to inhibit the Wnt/ß-catenin signaling pathway.

Expression and purification of the recombinant porcine NK-lysin in Pichia pastoris and observation of anticancer activity in vitro.

Natural killer (NK)-lysin, a broad-spectrum antimicrobial peptide, has antitumor and antibactericidal activities against both gram-positive and gram-negative bacteria. In this study the recombinant porcine NK-lysin was expressed and purified in the Pichia pastoris system, and then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to assess its anticancer activity in vitro. The results showed that the recombinant porcine NK-lysin possesses potent antitumor activity against the human hepatocellular carcinoma cell line SMMC-7721 in a time- and dose-dependent manner, but has negligible hemolysis activity against human erythrocytes. Scanning electronic microscopy was used to directly observe the ultrastructure of SMMC-7721 cells treated with NK-lysin; untreated cells showed lamellipodia and filopodia scattered with the cell surface, with good cell-cell contacts among neighboring cells. In contrast, treated tumor cells exhibited marked alterations in cell morphology, and cell-cell contacts disappeared among neighboring cells. Compared with the untreated tumor cells, the tumor cells treated with NK-lysin for 12 and 24 hr were suppressed for the expression of fascin 1. Thus, the recombinant porcine NK-lysin potentially could be developed as a therapeutic agent for inhibiting tumor growth.

Expression and purification of soluble porcine cystatin 11 in Pichia pastoris.

Cystatin 11 (CST11) belongs to the cystatin type 2 family of cysteine protease inhibitors and exhibits antimicrobial activity in vitro. In this study, we describe the expression and purification of recombinant porcine CST11 in the Pichia pastoris system. We then assess its antimicrobial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis by liquid growth inhibition assay. Kinetic studies indicate that the recombinant porcine CST11 has high potency against E. coli and S. aureus. Scanning electronic microscope analysis showed that CST11 might be targeting the bacterial membrane and, thus, could potentially be developed as a therapeutic agent for inhibiting microbe infection without the risk of antibiotic resistance.